Peroxidase enzymes such as horseradish peroxidase (HRP) are frequently used as markers or labels in enzyme-linked assays for biological molecules and other analytes of interest such as drugs, hormones, steroids and cancer markers. Detection of these enzymes can be achieved by the use of substrates which produce a detectable product. Chromogenic substrates such as o-phenylenediamine, ABTS or tetramethylbenzidine (TMB) produce a colored reaction product, fluorogenic substrates produce a fluorescent product, while chemiluminescent substrates produce light as the detectable product. Each of these methods can offer a safe, convenient and sensitive means to provide a quantitative measure of the amount of enzyme in a sample or of the amount of an enzyme-labeled analyte or labeled specific binding partner for an analyte.
a. Chemiluminescent Peroxidase Substrates
Applicant's U.S. Pat. Nos. 5,491,072, 5,523,212 and 5,593,845 disclose chemiluminescent N-alkylacridancarboxylic acid esters, thioesters and sulfonimides which produce light upon reaction with a peroxide and a peroxidase for use in detecting peroxidases and in assays. U.S. Pat. No. 5,545,834 describes the chemiluminescent reaction of spiroacridan compounds with hydrogen peroxide. The reaction is enhanced by the addition of horseradish peroxidase (HRP).
Various compounds of biological origin, collectively termed luciferins, are oxidized by a peroxidase (summarized in L. J Kricka and G. H. G. Thorpe, in Luminescence Immunoassay and Molecular Applications, K. Van Dyke and R. Van Dyke, eds., CRC Press, Boca Raton, 1990, pp. 77-98). In some instances, hydrogen peroxide is not utilized in which case the enzyme is functioning as an oxidase.
b. Fluorescent Peroxidase Substrates
U.S. Pat. No. 6,040,150 discloses an improved method of generating a fluorescent reporter compound from reaction of a fluorogenic substrate with a peroxidase. The method comprises using a phenolic compound as substrate including 3-(4-hydroxyphenyl)-propionic acid. Other phenolic fluorogenic substrates are disclosed in Zaitsu and Ohkura, Anal. Biochem., 109, 109-113 (1980) and include 2-(4-hydroxyphenyl)acetic acid, homovanillic acid and tyramine (Y. Li, et al., Anal. Chim. Acta, (340), 159-168, (1997)). Other art-known fluorogenic peroxidase substrates include o-phenylenediamine and N,N′-dicyanomethyl-o-phenylenediamine (Li, et al., Microchem. J., 53(4), 428-436 (1996)), amide and carbamate derivatives of p-aminophenol (M. Kawaguchi, et al., Bioluminescence and Chemiluminescence Perspectives for the 21st Century, A. Roda et al., Eds., Wiley & Sons, Chichester, pp 508-511, (1999)), 3,4-dihydro-2(1H)-quinoxalone and related derivatives (Li, et al., Anal. Chim. Acta, 340(1-3), 159-168 (1997)), reduced forms of fluorescein, rhodamine and other xanthine dyes and fluorinated derivatives of the latter (U.S. Pat. No. 6,162,931 and PCT Patent Publication WO99/01768).
c. Fluorescent Detection of Acridinium Labels
Acridinium esters and amides are known in the art of chemiluminescence. These compounds are generally used to label a substance to be detected in an assay. Detection by chemiluminescence comprises reaction of the label with a strongly alkaline hydrogen peroxide solution. A fluorometric assay of acridinium labeled compounds has also been described in which these same chemiluminescent compounds are measured by their inherent fluorescence (D. O. Shah and V. A. Salbilla, Proceedings of the 11th International Symposium on Bioluminescence and Chemiluminescence, J. F. Case et al., Eds., Wiley & Sons, Chichester, pp 235-238, (2001)).